T cells are the central organizers and effectors of the immune system and are responsible for effective immunity against pathogens and tumors as well as for keeping unresponsiveness (tolerance) against auto-antigens and harmless non-self antigens, such as food. To achieve this goal T cells are educated either during their early development in the thymus or later on in the periphery to acquire distinct effector functions, which can be stably inherited from a single cell to its progeny and in this way contribute to immunological memory. The type of effector function and specificity of a T cell population determines the outcome of the immune response and may therefore have high diagnostic or prognostic value for immune mediated diseases, infections or cancer. The type of T lymphocyte activation and differentiation into certain functional distinct populations is determined by co-stimulatory activation signals from antigen-presenting cells. Activation signals are represented by ligands for receptors of T lymphocytes. Said ligands are situated on the surface of the APCs, they are bound to the extracellular matrix or secreted by cells, as are the cytokines. However, in addition to antigen-specific activation by signals via the T cell receptor (TCR) and co-stimulating molecules, non-specific activation of T lymphocytes has also been described, e.g. via cytokines or lectins.
CD83 is preferentially expressed by DCs and serves as a marker for DC maturation (Zhou et al. 1992: J Immunol 149:735-742). Recently, it has been suggested that CD83 is involved in the regulation of B cell maturation, homeostasis and function (Breloer et al. 2007: Eur J Immunol 37:634-48; Kretschmer et al. 2007: PLoS 2:e755; Luthjy et al. 2008: Int Immunol 20:949-60; Kretschmer et al. 2009: 182:2827-34). Furthermore, results of several studies also noted important role of CD83 in T cell activation. Hirano et al have shown that membrane bound CD83 delivers a significant signal specifically supporting the expansion of newly primed naïve CD8 T cells (Hirano et al. 2006: Blood 107: 1528-1536). Consistent with this, lymphocytes from CD83−/−mice have shortened lifespans in vivo (Prazma et al. 2007: J Immunol 179:4550-4562). In addition, the transient overexpression of CD83 by immature and mature human DCs enhances their T cell stimulatory capacity (Aerts-Toegaert et al. 2007: Eur J Immunol 37:686-695).
In Kretschmer et al the authors provide evidence that activated murine T cells induce CD83 on B cells via CD40 engagement but independent of TCR/MHC binding and thus independent of antigen-specificity of T and B cells (Kretschmer et al. 2011: Immunology Letters 136; 221-227).
All those findings support the notion that CD83 has multiple confirmed functions in regulating immune system development and function.
The state of the art provides methods for the analysis of a T cell population after in vitro stimulation. Such methods are e.g. ELISPOT and intracellular cytokine staining (ICS). Visualizing cytokine producing cells with flow cytometry and ELISPOT technology has facilitated the accurate enumeration of antigen specific T cells producing different cytokines. However the dependency of stimulating the cells with antigen restricts these methods to a read out of one antigen per stimulation.
Another common technology is the p/MHC tetramer technology, which offers a great convenience to directly stain epitope specific T cells without need of any stimulation and has made it possible to perform direct phenotypic analysis of different T cells in the same culture. However this method is HLA type dependent, meaning that the HLA type of the sample T cells has to match the HLA type of the p/MHC tetramer conjugate used.
A novel method is the FTA T-helper assay to monitor murine CD4+ T cell-mediated B cell help in vivo using a multiplex high throughput assay. This assay utilizes a fluorescent target array (FTA), which is composed of spleenocytes labeled with numerous (>200) unique fluorescence signatures that can be delineated in a single recipient animal based on combination labeling with the three vital dye carboxyfluorescein diacetate succinimidyl ester (CFSE), CellTrace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD). By pulsing different B cell populations in a FTA with titrated amounts of cognate MHC-II binding peptides, CD4+ T cell help could be assessed by measuring induction of the B cell activation markers CD69 and CD44 by antibody labeling and flow cytometry (Quah et al. 2013: J Immunol Meth 387: 181-190). In another setting of the method also killing of CD8 T cells is analyzed. The disadvantages of the FTA are the restriction to a mouse in vivo model and the necessity of immunizing the animal prior the assay. To distinguish between CD8 and CD4 T cell responses the authors use MHC I or MHC II restricted peptides. By using multiple peptides one could not distinguish between a CD8 or CD4 derived B cell activation or lysis. Finally the FTA is not applicable to a human system as also the activation marker CD69 and CD44 are not specific for an antigen dependent cell-cell contact of B cells with T cells.
Therefore, the demand for an improved method to analyze simultaneously multiple human T cell populations in one sample remains still unsatisfied.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.